Method for producing multiple unsaturated fatty acids in plants

Abstract

The present invention relates to a method for the production of fatty acid esters which comprise unsaturated fatty acids with at least three double bonds, and to free unsaturated fatty acids with a content of at least 1% by weight based on the total fatty acids present in the plants, by expressing at least one nucleic acid sequence which encodes a polypeptide with Δ6-desaturase activity and at least one nucleic acid sequence which encodes a polypeptide with Δ6-elongase activity. Advantageously, these nucleic acid sequences can, if appropriate, be expressed in the transgenic plant together with a third nucleic acid sequence which encodes a polypeptide with Δ5-desaturase activity. The invention furthermore relates to the use of defined nucleic acid sequences which encode polypeptides with a Δ6-desaturase activity, Δ6-elongase activity or Δ5-desaturase activity selected from a group of nucleic acid sequences, and/or to the use of nucleic acid constructs comprising the abovementioned nucleic acid sequences.

1. A process for the production of compounds in transgenic plant cells, plants or parts thereof, wherein said compounds are comprised of general Formula I: 
       
         
           
           
               
               
           
         
         wherein R 1 ═—OH, coenzyme A (thioester), phosphatidylcholine, phosphatidylethanolamine, phoshatidylglycerol, diphosphatidylglycerol, phosphatidylserine, phosphadatidylinositol, sphingolipid, glycoshingolipid or a radical of general Formula II: 
       
       
         
           
           
               
               
           
         
         wherein R 2 =saturated or unsaturated C 20 -alkylcarbonyl-; and R 3 =saturated or unsaturated C 20 -alkylcarbonyl-; 
         wherein n=3, 4 or 6, m=3, 4 or 5; and p=0 or 3, and said compounds comprise at least 1% by weight of the total fatty acid content of said transgenic plants, which process comprises the following steps: 
         a) introducing, into a plant cell, plant or part thereof:
 i) at least one first nucleic acid sequence which encodes a polypeptide with an Δ6-desaturase activity; 
 ii) at least one second nucleic acid sequence which encodes a polypeptide with a Δ6-elongase activity; and 
 iii) a third nucleic acid sequence which encodes a polypeptide with a Δ5-desaturase activity; and 
 
         b) growing and harvesting the transgenic plant cell, plant or part thereof, 
         wherein said nucleic acid sequences are obtained from  Phaeodactylum tricornutum  or  Physcomitrella patens , and 
         wherein the at least one first nucleic acid sequence comprises the sequence of SEQ ID NO: 13, a nucleic acid sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 14, or a nucleic acid sequence encoding a polypeptide having at least 90% homology with the amino acid sequence of SEQ ID NO: 14 and having essentially the same enzymatic activity. 
       
     
     
       2. The process of  claim 1 , wherein R 2  and R 3  independently of one another are unsaturated C 20 -alkylcarbonyl- with one, two, three, four or five double bonds. 
     
     
       3. The process of  claim 1 , wherein the plant is an oil crop. 
     
     
       4. The process of  claim 1 , wherein the plant is selected from the group consisting of soya, peanut, oilseed rape, canola, linseed, evening primrose, verbascum, thistle, hazelnut, almond, macadamia, avocado, bay, wild roses, pumpkin/squash, pistachios, sesame, sunflower, safflower, borage, maize, poppy, mustard, hemp, castor-oil plant, olive, Calendula, Punica, oil palm, walnut and coconut. 
     
     
       5. The process of  claim 1 , wherein the compounds of general Formula I are obtained from the transgenic plants by pressing or extraction, and said compounds are in the form of oils, fats, lipids or free fatty acids. 
     
     
       6. The process of  claim 5 , wherein the oils, fats, lipids or free fatty acids are refined. 
     
     
       7. The process of  claim 1 , wherein saturated or unsaturated fatty acids present in the compounds are liberated. 
     
     
       8. The process of  claim 7 , wherein the saturated or unsaturated fatty acids are liberated by alkaline hydrolysis or enzymatic cleavage. 
     
     
       9. The process of  claim 1 , wherein the compounds comprise at least 5% by weight, of the total fatty acid content of the transgenic plants. 
     
     
       10. The process of  claim 1 , wherein the nucleic acid sequence that encodes the polypeptide with Δ6-elongase activity or Δ5-desaturase activity comprises a nucleic acid sequence selected from the group consisting of:
 a) a nucleic acid sequence comprising the sequence of SEQ ID NO: 3 or SEQ ID NO: 21, 
 b) a nucleic acid sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 11, SEQ ID NO: 18, or SEQ ID NO: 22, and 
 c) a nucleic acid sequence encoding a polypeptide having at least 90% homology with the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 22; wherein the polypeptide has essentially the same enzymatic activity. 
 
     
     
       11. The process of  claim 1 , wherein one or more of the first, second, and third nucleic acid sequences are linked with one or more regulatory signals in a nucleic acid construct. 
     
     
       12. The process of  claim 11 , wherein the nucleic acid construct comprises additional biosynthetic genes of fatty acid or lipid metabolism selected from the group consisting of acyl-CoA dehydrogenase(s), acyl-ACP [=acyl carrier protein] desaturase(s), acyl-ACP thioesterase(s), fatty acid acyl transferase(s), fatty acid synthase(s), fatty acid hydroxylase(s), acetyl-coenzyme A carboxylase(s), acyl-coenzyme A oxidase(s), fatty acid desaturase(s), fatty acid acetylenases, lipoxygenases, triacylglycerol lipases, allene oxide synthases, hydroperoxide lyases fatty acid elongase(s) and combinations thereof. 
     
     
       13. The process of  claim 1 , wherein the first, second and third nucleic acid sequences are stably integrated in the plant. 
     
     
       14. The process of  claim 1 , wherein the plant or part thereof comprises plant tissues, plant organs, plant leaves, plant roots, plant stems, intact plants, plant tubers, plant seeds, or cellular parts of any of the preceding. 
     
     
       15. The process of  claim 5 , wherein the pressing or the extraction is performed without supplying heat. 
     
     
       16. The process of  claim 1 , wherein the at least one second nucleic acid sequence comprises:
 the sequence of SEQ ID NO: 3, 
 a nucleic acid sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 4, or 
 a nucleic acid sequence encoding a polypeptide having at least 90% homology with the amino acid sequence of SEQ ID NO: 4, and having essentially the same enzymatic activity. 
 
     
     
       17. The process of  claim 1 , wherein the third nucleic acid sequence comprises:
 the sequence of SEQ ID NO: 21, 
 a nucleic acid sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 22, or 
 a nucleic acid sequence encoding a polypeptide having at least 90% homology with the amino acid sequence of SEQ ID NO: 22 and having essentially the same enzymatic activity. 
 
     
     
       18. The process of  claim 11 , wherein the first, second and third nucleic acid sequences are linked with one or more regulatory signals in said nucleic acid construct. 
     
     
       19. The process of  claim 1 , wherein the plant is a dicot. 
     
     
       20. The process of  claim 1 , wherein the plant is tobacco or linseed. 
     
     
       21. The process of  claim 1 , wherein the at least one second nucleic acid sequence comprises the sequence of SEQ ID NO: 3, a nucleic acid sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 4, or a nucleic acid sequence encoding a polypeptide having at least 90% homology with the amino acid sequence of SEQ ID NO: 4 and having essentially the same enzymatic activity, and the third nucleic acid sequence comprises the sequence of SEQ ID NO: 21, a nucleic acid sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 22, or a nucleic acid sequence encoding a polypeptide having at least 90% homology with the amino acid sequence of SEQ ID NO: 22 and having essentially the same enzymatic activity.